CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF's effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (JV) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruch's membrane complex and provide protection against neurodegenerative disease

Gene expression of CNTF, CT1, OsM, LIF and their receptors in hfRPE.

<p>Relative amounts of messenger RNA were quantified using real time PCR. All data normalized to GAPDH = 10⁶. Experiments were performed in triplicate using cells from four different donors.</p

CFTRinh-172 inhibits CNTF-induced fluid transport across RPE.

<p><i>J</i>_V is plotted as a function of time in the top trace and net fluid absorption (apical to basal bath) is indicated by positive values; TEP (-) and R_T (Δ) are plotted as function of time in the lower traces. For each set of experiments, left- hand panels (<b>A, C, E</b>), summary bar graphs are shown on the right-hand side (<b>B, D, F,</b> respectively) <b>A.</b> Addition of CNTF to the apical and basal bath increased <i>J</i>_V across monolayer of hfRPE; subsequent addition of CFTRinh-172 decreased <i>J</i>_V back to the baseline levels (n = 5). <b>C.</b> Pretreatment with JAK inhibitor I (5 µM) significantly inhibited the effect of CNTF on <i>J</i>_V increase (n = 3). <b>E.</b> Subsequent addition of JAK inhibitor I decreased <i>J</i>_V induced by CNTF (n = 3). * <i>P</i><0.05, ** <i>P</i><0.01 compared to each other.</p

Dose responses of CNTF, CT1, and OsM - induced hfRPE proliferation.

<p>CNTF showed no significant effect on hfRPE proliferation, while CT1 significantly increased cell proliferation by 25% (50–100 ng/ml). In contrast, OsM produced a monotonic increase in its inhibitory effect on hfRPE proliferation between 1 and 100 ng/ml. There was a significant difference in inhibition between 1 and 10 ng/ml (<i>P</i> = 0.02) and between 10 and 100 ng/ml (<i>P</i> = 0.001). 5% FBS was used as positive control. (Summary data from two experiments using cells from different donors; * <i>P</i><0.05, *** <i>P</i><0.001 compared to 0% FBS negative control).</p

Schematic diagram of CNTF, CT1 and OsM cytokine receptor complexes.

<p>The cytokines are depicted schematically as grey circles. Signal transducing-receptor subunits are light blue (LIFRβ and OsMRβ) or dark blue (gp130). α-receptor subunits are shown in light grey.</p

Constitutive expression of CNTF, CT1, OsM receptors in human RPE.

<p>15 µg of enriched membrane proteins were electrophoresed and labeled with corresponding antibodies. Antibody specific bands (see arrows) for CNTFRα, LIFRβ, gp130 and OsMRβ are detected at approximately 41, 190, 130, and 200 kDa, respectively. <b>Panel A</b> (CNTFRα) - Lane 1: Magic Mark™ XP Western protein Standard; Lane 2: Brain (Human) membrane lysate-adult normal tissue; Lane 3: hfRPE membrane extract. <b>Panel B</b> (LIFRβ) - Lane 1: Magic Mark™ XP Western protein Standard; Lane 2: hfRPE membrane extract. <b>Panel C</b> (gp130) - Lane 1: HiMark™ Molecular Weight (HMW) Standard; Lane 2: hfRPE membrane extract; Lane 3: Magic Mark™ XP Western protein Standard; Lane 4: Hela whole cell lysate; Lane 5: NIH3T3 whole cell lysate. <b>Panel D</b> (OsMRβ) - Lane 1: HiMark™ Molecular Weight (HMW) Standard; Lane 2: hfRPE membrane extract; Lane 3: Magic Mark™ XP Western protein Standard.</p

Schematic diagram of fluid transport chamber.

<p>Top part of figure shows internal chamber design consisting of two identical hemichambers. Each hemichamber has a voltage-sensing electrode, three current-passing electrodes, and a thermistor. Hemichamber A contains media bathing the apical or retinal surface; hemichamber B contains solution bathing the basal or choroidal surface. Surface of each bathing solution is covered with hexadecane to eliminate evaporation. A capacitance probe is immersed in the hexadecane in one hemichamber. Volume change is detected by changes of distance between probe and bathing media surface. Solutions are exchanged by gravity flow from reservoirs through inflow valves at the bottom and suction from the surface on top. A thin Kel-F disc (dark horizontal line) is used to isolate probe tip from media and avoid short circuit. RPE monolayer (E) is mounted between two circular Kel-F discs with O-rings (shown in lower left corner of the figure). This chip is mounted between two water-jacketed Kel-F blocks in main chamber located in a modified CO₂ incubator (5% CO₂, 36.5°C) for fluid transport (<i>J</i>v) measurements.</p

Immunofluorescence localization of CNTF, CT1, OsM receptors on hfRPE.

<p>Central part of each panel is an <i>enface</i> view of cell culture monolayer, shown as a single optical section obtained from a Z-stack. Nuclei were stained with DAPI (blue) and ZO-1 tight junction marker stained green. Images of the cross section through the Z-plane are shown at the top of each panel. CNTFRα (A, red), LIFRβ (B, red), gp130 (C, red) and OsMRβ (D, red) were detected mainly on the apical membrane of confluent monolayer of hfRPE.</p